Mannich bases 2a-f derived from 3,4-dimethylphenol (1), formaldehyde and different amines are prepared and subjected to spectral (IR, (1)H and (13)C NMR) and elemental analyses. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with 1 and synthesized Mannich bases 2a-f and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. In relation to hydratase and esterase activities of the half maximal inhibitory concentration (IC(50)) and the inhibition equilibrium constants (K(i))values were determined. Only two compounds (2a and 2e)exhibit weak hCA II inhibitory effects on esterase activity. IC(50) and Ki values for 2a and 2e with respect to esterase activity of hCA II are0.88 × 10(3) and 6.3-7.6 µM and 0.44 × 10(3) and 19.0-96.4 µM,respectively. On the contrary, compounds 2b and 2d might be used as CA activators due to increasing esterase activity of hCA I and hCA II isozymes.